New mediators for the enzyme laccase: Mechanistic features and selectivity in the oxidation of non-phenolic substrates

Article abstract of DOI:10.1039/b507657a

New mediators of laccase have been comparatively evaluated and ranked towards the benchmark aerobic oxidation p-MeO-benzyl alcohol. The mechanism of oxidation of this non-phenolic substrate by each mediator, which is initially oxidised by laccase to the Med_oxform, has been assessed among three alternatives. The latter make the phenoloxidise laccase competent for the indirect oxidation of non-phenolic (and thus 'unnatural') substrates. Experimental characterisation of the mediators, by means of spectrophotometric, electrochemical and thermochemical survey, is reported. Clear-cut evidence for the formation of a benzyl radical intermediate in the oxidation of a particular benzyl alcohol with laccase and a 2 bonds on the lefthand side signN-OH mediator is attained by means of a trapping experiment. The selectivity of the laccase-catalysed oxidation of two competing lignin and polysaccharide model compounds has been assessed by using the highly proficient 4-MeO-HPI mediator, and found very high in favour of the former model. This evidence is in keeping with the operation of a radical hydrogen-abstraction process that efficiently cleaves the benzylic rather than the aliphatic C-H bond of the two models. Significant is the finding that catechol, i.e., a model of recurring phenolic structures in lignin, once oxidised to aryloxyl radical by laccase is capable to mediate a radical oxidation of non-phenolic compounds. This supports a fully-fledged role of laccase as a delignifying enzyme in nature by way of no other mediators than the very phenolic groups of lignin. Finally, an evaluation of the dissociation energy of the NO H bond of HBT, which is not accessible experimentally, is provided by the use of a thermochemical cycle and theoretical calculations. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2005.

Full text of DOI:10.1039/b507657a

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P A P E R
New mediators for the enzyme laccase: mechanistic features and
selectivity in the oxidation of non-phenolic substrates
Paola Astolfi,a Paolo Brandi,b Carlo Galli,*b Patrizia Gentili,*b Maria Francesca Gerini,b
Lucedio Grecia and Osvaldo Lanzalungab
a
`
Dipartimento di Scienze dei Materiali e della Terra, Universita di Ancona, 60131 Ancona,
Italy
Dipartimento di Chimica, Universita ‘La Sapienza’, and IMC-CNR Sezione Meccanismi di
b
`
Reazione, 00185 Roma, Italy. E-mail: carlo.galli@uniroma1.it.; Fax: þ39 06 490421
Received (in Montpellier, France) 31st May 2005, Accepted 15th July 2005
First published as an Advance Article on the web 5th August 2005
New mediators of laccase have been comparatively evaluated and ranked towards the benchmark aerobic
oxidation of p-MeO-benzyl alcohol. The mechanism of oxidation of this non-phenolic substrate by each
mediator, which is initially oxidised by laccase to the Medox form, has been assessed among three
alternatives. The latter make the phenoloxidise laccase competent for the indirect oxidation of non-phenolic
(and thus ‘unnatural’) substrates. Experimental characterisation of the mediators, by means of
spectrophotometric, electrochemical and thermochemical survey, is reported. Clear-cut evidence for the
formation of a benzyl radical intermediate in the oxidation of a particular benzyl alcohol with laccase and a
{N–OH mediator is attained by means of a trapping experiment. The selectivity of the laccase-catalysed
oxidation of two competing lignin and polysaccharide model compounds has been assessed by using the
highly proficient 4-MeO-HPI mediator, and found very high in favour of the former model. This evidence is
in keeping with the operation of a radical hydrogen-abstraction process that efficiently cleaves the benzylic
rather than the aliphatic C–H bond of the two models. Significant is the finding that catechol, i.e., a model
of recurring phenolic structures in lignin, once oxidised to aryloxyl radical by laccase is capable to mediate a
radical oxidation of non-phenolic compounds. This supports a fully-fledged role of laccase as a delignifying
enzyme in nature by way of no other mediators than the very phenolic groups of lignin. Finally, an
evaluation of the dissociation energy of the NO–H bond of HBT, which is not accessible experimentally, is
provided by the use of a thermochemical cycle and theoretical calculations.
other enzyme involved in the biodelignification of rotten wood
Introduction
is laccase, a family of multicopper oxidases endowed with
redox potential in the 0.5–0.8 V/NHE range.4,7,8 Due to the
lower redox potential,9 laccase can oxidise monoelectronically
only the phenolic groups of lignin (phenoloxidase activity),4,10
but these represent less than 20% of all the functional groups
of the polymer.11 The benzyl alcohol and ether groups, sum-
ming up to about 70% of the residues but being more resistant
to monoelectronic oxidation (redox potentials 4 1.5 V/NHE),
cannot be oxidised by laccase directly. Use of appropriate
redox metabolites, or of purposedly added compounds, enables
laccase to oxidise indirectly even non-phenolic groups in a
catalytic cycle.1,4,5,12 Following monoelectronic interaction
with the enzyme, the oxidised messenger (Medox) oxidises
‘unnatural’ non-phenolic substrates according to mechanisms
not available to laccase, as sketched in Scheme 1 in very general
terms. In this way the messenger expands the oxidation ability
of the enzyme, and more specifically fulfils the role of a
‘mediator’.1,13
Several laccase/mediator systems have been investigated and
found valuable in the aerobic oxidation of non-phenolic lignin
models (such as benzyl alcohols),14 and of lignin itself from
industrial wood pulp.15–17 Our contribution to this field has
been to provide a consistent way to evaluate and compare the
efficiency of a number of laccase mediators: the oxidation of a
specific non-phenolic substrate, i.e. 4-methoxybenzyl alcohol
(viz. p-anisyl alcohol), under strictly reproducible conditions
represents the benchmark reaction over which the merits
of several mediators can be assessed unambiguously.14 This
The world of biochemistry offers many examples of the role of
a messenger. For example, the RNA-messenger delivers the
genetic information to the proteins factory embodied by the
ribosome, whereas cyclic-AMP promotes the metabolism of
lipids and sugars. A messenger solves communication needs,
but it may also act as a catalyst.1 Peculiarities of the role may
require turning the name messenger into that of mediator: this
becomes apparent in the field of enzymatic oxidations and, in
particular, in the degradation of wood. Basidiomycete ‘white-
rot’ fungi perform the delignification of dead wood through a
complex but efficient strategy, requiring specialised extracellu-
lar enzymes.2–4 The enzymes oxidise lignin in woody tissue, in
order to make cellulose available to the metabolism of fungi.
Lignin, however, the largest alkylaromatic polymer in nature,
is so structurally complex that a direct interaction with the
active site of an enzyme is sterically unfeasible. The problem is
solved by appropriate messengers.4,5 These low-molecular-
weight metabolites, once oxidised by the enzymes, sneak away
into the woody fibres and deliver oxidative equivalents to
appropriate functional groups of the lignin polymer. For
example lignin peroxidase,6 a heme enzyme endowed with a
redox potential of 1.3–1.4 V/NHE and with a small active site,
oxidises the metabolite veratryl alcohol (i.e., 3,4-dimethoxy-
benzyl alcohol; E1ox 1.36 V) to its radical cation by electron
abstraction. The latter, in turn, removes electrons from lignin,
causing its degradative oxidation and consequently solving the
communication problems between enzyme and substrate. An-
T h i s j o u r n a l i s & T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 5
1308
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7

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necessary here. Direct hydrogen-atom transfer (viz., the HAT
mechanism)13,14 from the benzylic C–H bond of p-anisyl
alcohol is precluded to all of these {N–Od compounds on
enthalpic grounds (Scheme 3).19,20 In fact, the {NO–H bond
that ought to be formed is weaker than the benzylic C–H bond
to break (ca. 83 kcal molꢀ1),19a as the BDENOꢀH values (r72
Scheme 1 The oxidation cycle of a laccase/mediator system towards
non-phenolic substrates.
kcal mol )
ꢀ1 19b of the parent hydroxylamines indicate (Table 1).
The reason why the aminoxyl radical TEMPO (entry 5)13,14
performs instead so well as mediator is that its monoelectronic
approach has further enabled us to characterise three different
oxidation routes that the mediators of laccase can undertake
depending on their structure.1,13
oxidation to the oxoammonium form ({N O1) occurs at a
Q
redox potential (ca. 0.7 V/NHE)14 that matches the one of
Trametes villosa laccase.9 Thus the ionic mechanism of oxida-
tion (Scheme 4), where laccase recycles reduced-TEMPO to the
active species by electron-transfer oxidation,13,14,23 takes over.
In the present paper we (i) report on new mediators, inclu-
ding a few that the recent literature has brought to our
attention, (ii) evaluate their efficiency and merits with respect
to those already investigated, and (iii) characterise their oper-
ating mechanism. Relevant redox, spectrophotometric and
thermochemical data of the mediators have been acquired,
aiming at finding a relationship between reactivity features and
structure. The selectivity of one of the most proficient media-
tors of laccase has been assessed in the competitive oxidation of
a lignin model with a polysaccharide model compound. Final-
ly, the benzyl radical intermediate, resulting from the oxidation
of a suitable benzyl alcohol with a laccase/mediator system, has
been intercepted for the first time.
This route via the {N O1 ion can be undertaken with
Q
equal success by 4-substituted TEMPO derivatives (cf. entry
6)14 or by the 5-membered analogue PROXYL (entry 8), in
view of the comparable {N O1/{N–Od redox values.
Q
Accordingly, in the oxidation of a-monodeuteriated p-MeO-
benzyl alcohol with laccase/PROXYL we give here a value of
the intramolecular kinetic isotope effect (kH/kD ¼ 2.4) equal to
that (kH/kD ¼ 2.2)1 reported for the oxidation by laccase/
TEMPO. This is in keeping with the operation of the ionic
oxidation mechanism, where deprotonation of the TEMPO–
alcohol (or PROXYL–alcohol) adduct en route to the oxida-
tion product is rate determining with electron-donor substi-
tuted benzyl alcohols.1,23 The ionic route has precedents
Results and discussion
whenever TEMPO is oxidised to the {N O1 form by
Q
Determination of the efficiency of new mediators
suitable oxidants (e.g., PhI(OAc)2; cupric salts; Mn–Co salts)24
other than the enzyme laccase. In contrast, a radical hydrido-
metal mechanism is documented when TEMPO is used in
combination with RuCl2(PPh3)3, and in fact a different and
larger kinetic isotope effect value (kH/kD ¼ 5.1) is reported for
a-monodeuteriated benzyl alcohol.25
The aerobic oxidation of p-anisyl alcohol, catalysed by laccase
from Trametes villosa, is taken up in order to rank the
efficiency of the new mediators.14 The reaction is run at room
temperature for a fixed time span (24 h), in buffered water
solution (3 mL; pH 5) previously purged with O2 (Scheme 2).
The experimental conditions adopted, namely, [p-anisyl
alcohol] 20 mM, [mediator] 6 mM, with 3 U mLꢀ1 of laccase,
are such as to comply with the ‘ideal’ scheme of a mediated
oxidation (Scheme 1) where the mediator, in deficiency with
respect to the substrate, turns over between its natural and
oxidised (Medox) states, due to the intervention of laccase and
O2, while carrying out the oxidation of the non-phenolic
substrate in a catalytic cycle.14,18
The ionic oxidation route available to TEMPO is instead
precluded to the aminoxyl radicals in entries 1, 3 and 4, because
conversion to their corresponding {N O1 ion takes place at
Q
a redox potential higher (c1.1 V;22a,f for entry 3 we obtain
here 1.2 V/NHE) and not attainable to laccase. The lack of
efficiency with (t-Bu)2N–Od (entry 2), which is an open-chain
structural counterpart of TEMPO, and has a redox potential
(Ep 0.6 V)22b,g that could enable conversion into the {N O1
Q
ion by laccase, would seem puzzling. However, the instability
of the oxidised form of (t-Bu)2N–Od has been already discus-
sed,22g,h and explains the irreversible electrochemical peak
determined, as opposed to the case of TEMPO. Finally, the
commercially available polymer-bound TEMPO comes out
inactive as a laccase mediator (entry 7): it is indeed covalently
linked to the polymeric support through its N–O bond, and
therefore conversion into the oxoammonium ion, and opera-
tion of the ionic oxidation route, are precluded.
In Table 1 the conversion into product, investigated with 15
new potential mediators, is given and compared with that of
other mediators already examined under these conditions.14
No other oxidation products besides p-anisaldehyde appear,
the rest of mass balance being the recovered p-anisyl alcohol.
Significant thermochemical data (bond dissociation energy,
BDE) of the mediators are listed in the Table, either experi-
mental or calculated ones.19,20 The redox potential of some
mediators has been determined by cyclic voltammetry here,
while already available values for other mediators are
added.14,21,22 On the basis of the data in Table 1, we can recog-
nise three groups of mediators according to their behaviour
and structure, and a more satisfactory mechanistic assessment
of the mediation phenomenon now becomes possible.
The {N–OH mediators
Leading examples in this class are violuric acid (VLA), N-
hydroxyphthalimide (HPI) and 1-hydroxybenzotriazole (HBT)
(entries 14–16), which perform with laccase well in the oxida-
tion of non-phenolics via the HAT mechanism (cf. Scheme
3).1,13,14 In general terms, monoelectronic oxidation of {N–
OH by laccase generates the {N–OHd1 species that deproto-
nates to the aminoxyl radical ({N–Od) as the reactive inter-
mediate. However, at the pH of the benchmark oxidation
reaction (i.e., pH 5) many {N–OH derivatives are already
present as {N–Oꢀ, and directly converted to {N–Od by
laccase. Independent monoelectronic generation of the ami-
noxyl radicals from both HBT and HPI by the use of suitable
oxidants, such as CeIV or Pb(OAc)4,26 is reported and confirms
this point. The experimental BDENO–H value (88.1 kcal
The {N–Od species
The negligible oxidation of the substrate documented in entries
1–4 (Table 1) points out that the aminoxyl radicals are not
efficient mediators of laccase, with the exception of TEMPO
and its cognates (entries 5,6,8), and a twofold explanation is
molꢀ1 19b
of HPI supports an exothermic HAT process by its
)
Scheme 2 The benchmark aerobic oxidation of p-anisyl alcohol by
laccase/mediator/O2.
corresponding {N–Od species. For entries 12 and 13, avail-
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7
1309

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Table 1 Benchmark aerobic oxidation of p-anisyl alcohol with laccase/mediator systems (Scheme 2) in 3 mL buffered water solution (0.1 M sodium
citrate, pH 5) saturated with O2.a Yields of the oxidation product are determined by GC after a 24 h reaction time at room temperature. Redox and
thermochemical data of the mediators are added, either determined here or already available
c
Yield of
p-anisaldehyde (%)b
BDENO–H /kcal molꢀ1
(from ref. 19a)
E1/V vs. NHE
No.
1
Mediator
in H2O14,21,22
Fremy salt
o1
—
1.36 (ref. 22f)
2
3
Di-tert-butylnitroxide
1
r69
0.6 (Ep value) (refs. 22b,b)
INDO
0d
71 (ref. 19b)
1.2 (in MeCN)
4
5
o1
ca. 71 (cf. entry 3).
ca. 1.2 (cf. entry 3)
TEMPO
99e
69.6
0.73 (0.92 in MeCN)
6
7
4-OH-TEMPO
98e
0
72
—
0.85 (ref. 22c)
TEMPO-polymer bound
—
8
9
PROXYL
83
22
70
—
1.1 (in MeCN)
3-MPO
41.1
10
11
12
ABTS
21e
19
7
—
0.69 and 1.1 (ref. 14)
HBO
Z 80 (cf. ref. 19b)
78–79 (cf. ref. 19b)
1.1 (E p value; E1 1.2 in MeCN)
0.8 (also in MeCN)
13
14
15
16
NHA
VLA
HPI
54f
85
80
0.8 (ref. 21)
ca. 80–85 (cf. ref. 19c,d)
88.1 (ref. 19b)
(cf. ref. 20)
0.92 (0.95 in MeCN)
1.09 (0.92 in MeCN)
1.08 (0.97 in MeCN)
70e
76e
HBT
17
18
TFBT
33
38
(cf. ref. 20)
1.11 (in MeCN)
1.14 (in MeCN)
HOAT
—
19
NBI
11
80 (in ref. 19b)
41.2 (this work and ref. 22i)
1310
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7

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Table 1 (continued )
c
Yield of
p-anisaldehyde (%)b
BDENO–H /kcal molꢀ1
(from ref. 19a)
E1/V vs. NHE
No.
Mediator
in H2O14,21,22
20
21
HAA
2
—
—
—
—
2-NH2-purine
14
22
Phenol red
20g
ca. 85–90 (as BDE(O–H)
0.8 (ref. 22e)
23
24
25
26
Cresol red
20g
55g
5g
See above
See above
See above
See above
—
0.7 (ref. 22e)
Dichlorophenol red
Phenolphthalein
Catechol (1,2-dihydroxybenzene)
—
—
15
27
Galvinoxyl
o1
79
—
a
b
Conditions: [p-anisyl alcohol] 20 mM, [mediator] 6 mM, laccase 3 U mLꢀ1
of mass balance being recovered starting material; no other products besides p-anisaldehyde are detected. In buffered water or in MeCN with Py
.
Yields (ꢁ2%) are reckoned vs. the molar amount of alcohol, the rest
c
(see Experimental). The E1 data pertain to the {N O1/{N–Od, Rd /R or ArOHd1/ArOH couples. From ref. 23a. From ref. 14. From
ref. 21. From ref. 22e.
1
d
e
f
Q
g
able BDENOꢀH values are analogously Z 79 kcal molꢀ1 19,20
,
literature evidence for structurally similar compounds (E1
values Z 1.3 V),22i and consequently hampers the generation
of the Medox form.
and compatible with mediation efficiencies ranging from mod-
erate to good (Table 1). In this respect, the lower efficiency
displayed (in entry 12) by the cyclic counterpart of the profi-
cient mediator N-hydroxyacetanilide (NHA, entry 13),21 could
be due to the slightly lower BDENO–H value.
Large values of the kinetic isotope effect (kH/kD in the 5.2–
6.4 range)1,13,21 are obtained for this group of mediators on
using a-deuteriated benzyl alcohols, as expected for a rate-
determining H-abstraction mechanism of oxidation (Scheme
3). The operation of the ionic route documented for TEMPO
(Scheme 4)13,23 would be an unlikely alternative to the HAT
route of the {N–OH mediators, because further oxidation of
Commercially available aryl-substituted HBTs, bearing the
electron-withdrawing CF3– (entry 17) or aza-groups (entry 18),
show a mediation efficiency lower than HBT itself (entry 16).
Their redox potentials have been determined here by cyclic
voltammetry, and found higher in value than that of HBT.
As a consequence, oxidation by laccase to the corresponding
{N–Od form is relatively more difficult and consistent with a
lower efficiency as mediators. Furthermore, the aryl-substitu-
ent is likely also to affect the BDENO–H value and, in the H-
abstraction step from the substrate, a lower BDENO–H value
would be detrimental to the efficiency of the HAT route.20
Unfortunately, BDENO–H values could not be determined for
the HBTs family, due to the short life-time of the involved
{N–Od species that prevents the use of the EPR equilibration
method.19b,c However, this point could be investigated experi-
mentally within the family of the aryl-substituted HPIs (see
below); moreover, use of a thermochemical cycle (and theore-
tical calculations) allows us to extract a BDENO–H value for
HBT and even for other {N–OH mediators (vide infra).
Another mediator (entry 19; N-t-Bu-3,5-dinitrobenzohy-
droxamic acid, NBI) of this group shows a modest efficiency
in spite of a BDENO–H value that could be adequate.19b
However, the redox potential that we attempted to measure
appears to be beyond the reach of laccase, in keeping with
their {N–Od intermediate to the corresponding {N O1
Q
species takes place at a E1 value high (41.3 V) and inaccessible
to laccase.
To this group belongs 3-hydroxybenzotriazin-4-one (HBO,
entry 11) that was suggested to be a valuable mediator of
laccase.20,22d The objective comparison of efficiency through
the benchmark oxidation (Table 1) sets instead HBO on
a modest level among the {N–OH mediators. Anyhow, its
{N–Od form (dubbed here BONO) has been independently
generated by us with a stoichiometric amount of the mono-
electronic oxidant cerium(IV)ammonium nitrate, CAN, and
observed spectrophotometrically in MeCN solution (Fig. 1).
It presents UV spectral features (lmax ¼ 524 nm, e ¼ 1100 Mꢀ1
cmꢀ1) similar to those of the currently investigated aminoxyl
radicals from HBT (i.e., BTNO) and HPI (i.e., PINO),26 and a
Scheme 3 The thermochemical scheme of the radical H-atom transfer
(HAT) mechanism between a {N–Od species and a C–H donor
substrate.
Scheme 4 The ionic mechanism of oxidation of benzyl alcohols by an
oxoammonium ion.
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7
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tron-rich substrates, has not been reported yet.30 Anyhow,
Table 1 points out that the mediation efficiency of laccase/
ABTS towards p-anisyl alcohol is moderate (entry 10).14 A new
mediator, i.e., 1-(30-sulfophenyl)-3-methylpyrazolone-5 (3-
MPO), has recently been described to be comparable in both
mediation efficiency and oxidation mechanism to ABTS.31 This
is confirmed by the result of our benchmark oxidation (entry
9). Furthermore, the addition of a stoichiometric amount of
the CeIV oxidant to a water solution of 3-MPO in a spectro-
photometric cuvette gives rise to a broad absorption band
having lmax ¼ 314 nm and e ¼ 1100 Mꢀ1 cmꢀ1. It sponta-
neously decays with a half-life of only 8 s, which certainly
undermines the value of 3-MPO as a potential diffusible
mediator of laccase.5,12 An attempt to determine the redox
potential of 3-MPO has been made by cyclic voltammetry but
without success: no distinct oxidation wave appears on scan-
ning the potential up to 1.1–1.2 V.
Natural mediators
In keeping with the concept of redox mediators delineated in
the Introduction,1,4,5,12 natural metabolites of laccase have
been actively searched, but not many have been found and
moreover these give contrasting results.18,32 Phenol or aniline
derivatives were at times indicated as potential natural media-
tors of laccase, two exemplary cases being 3-hydroxyan-
thranylic acid (HAA) and 2-aminopurine;33,34 however, our
benchmark oxidation gives negligible or modest mediation
efficiency with them (entries 20 and 21). The dimerisation
reaction of HAA to cinnabarinic acid ensuing oxidation by
laccase is indeed well established,32 and proceeds through a
typical phenoxyl–radical coupling; the dimer is not active in
any oxidation mechanism of non-phenolics.32 As a matter of
fact, reports of successful mediation with those ‘natural’
compounds relied on peculiar mediator-to-substrate molar
ratios as high as 40 or even 200.34 At those high concentrations
of ‘mediator’ it is conceivable that any degradation route of the
Medox species, taking place in the reaction medium and leading
to side-products of unknown structure, might concur to the
onset of oxidation routes of unidentified nature.18,30c
In contrast, we reported that Phenol Red and Dichlorophe-
nol Red mediate laccase efficiently in the oxidation of p-anisyl
alcohol (entries 22–24).22e This was attributed (i) to the low
pKa value of these phenolic compounds (i.e., 7 or below), that
makes easy both their conversion into the ArOꢀ form, as well
as the subsequent monoelectronic oxidation to ArOd by lac-
case, but also (ii) to their fair stability as aryloxyl radicals. The
ArOd radical in fact must be sufficiently ‘stable’ to enable the
H-removal from non-phenolics before disappearing in unde-
sired phenol-coupling routes.22e The BDEO–H of phenol deri-
vatives appears appropriate to a moderately exothermic H-
abstraction route as in Scheme 3,19a in keeping with the case of
the {N–Od intermediates. Phenolphthalein (entry 25) is less
acid (pKa 8) than the cognate compounds of entries 22–24, and
this could explain its lower efficiency at the pH of the bench-
mark reaction. Finally galvinoxyl (entry 27), although a per-
sistent phenoxyl radical, is endowed with a lower BDEO–H that
makes the H-abstraction route endothermic.19a It also suffers
from the steric hindrance of two t-Bu groups ortho to the
phenoxyl radical center, possibly impairing the interaction with
potential substrates. Catechol (entry 26) is instead a newly
found active mediator, and this has an important implication
because the ortho-diphenolic moiety is a recurring structural
feature in lignin,11 and benzenediols are typical substrates of
laccase.4,8 The concluding message is that phenolic derivatives,
including suitable phenolic moieties of lignin, once oxidised by
laccase to phenoxyl radicals, can behave as mediators for the
radical oxidation of non-phenolic groups of lignin, provided
that they are both easily oxidisable and sufficiently stable as
phenoxyl radicals to enable the occurrence of the H-abstrac-
Fig. 1 UV-Vis spectrum of BONO in MeCN: (a) immediately after
the generation with CAN (curve m), (b) after 2 h (curve ’).
kinetic investigation of its H-abstraction proficiency is
planned. The formation of the BONO radical is followed by
its spontaneous decay, which is faster (first half-life: 400 s) than
that of PINO (first half-life: 7900 s)26a,b but slower than that of
BTNO (110 s).26c
The redox potential of HBO has been determined by cyclic
voltammetry (Table 1). An EPR spectrum of BONO has been
acquired in MeCN, from reaction of HBO with CAN (Fig. 2).
Redox mediators
Among the artificial mediators of laccase, ABTS (2,20-azino-
bis(3-ethylbenzthiazoline-6-sulfonate) was the first to be em-
ployed.1,4,5,7,12,14,27 It undergoes one-electron oxidation to the
relatively stable (half-life of 90 min) blue-coloured radical
1
cation ABTSd (lmax ¼ 420 nm, e ¼ 3.5 ꢂ 104 Mꢀ1 cmꢀ1),28
at a redox potential (0.69 V)14 that almost matches that of
laccase,9 so to provide the standard assay of the activity of this
1
enzyme.29 Further oxidation of ABTSd to dication ABTS21
occurs electrochemically at higher potential (1.1 V),5,14,28 but
this red-coloured species is less stable.28 The laccase/ABTS
system has been reported to oxidise non-phenolic com-
pounds,4,5,7,12,27 but a precise assessment of the structure of
the intervening Medox form (whether ABTSd1 or ABTS21), as
responsible for the monoelectronic oxidation of suitably elec-
Fig. 2 Experimental EPR spectrum from HBO with CAN in MeCN
(upper) and its computer simulation (lower); EPR data: a(N) ¼ 1.38 G,
a(N) 2.74 G, a(N) ¼ 4.60 G; a(H) ¼ 0.53 G, a(H) 1.38 G; g ¼ 2.0062.
1312
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7

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tion route. This conclusion, and the case of catechol in
particular, supports a proficient delignifying role for the phe-
noloxidase laccase in nature even in the absence of added
‘foreign’ mediators.1,10,22e,35
Derivatives of HPI in reaction with laccase
Among the {N–OH mediators, HPI is of particular synthetic
interest because, in combination with co-catalysts like
Co(OAc)2 or Co(acac)2,36,37 it efficiently catalyses the oxida-
tion of a large variety of organic compounds under mild
conditions, at moderate oxygen pressure and temperature.
The above mentioned phthalimide-N-oxyl radical (PINO) is
the active oxidant, which oxidises the substrate by the radical
H-abstraction route, regenerating HPI.19b,26,36–38 In this con-
text we considered that it would be worthwhile understanding
how the introduction of substituents in HPI can affect the
mediation efficiency with laccase. To this purpose, several aryl-
substituted HPI’s containing either electron-withdrawing (4-
MeOCO, 3-F) or electron-donating groups (4-Me, 4-MeO)
have been synthesised and investigated.26b
Scheme 5 Mechanism of oxidation of p-anisyl alcohol promoted by
laccase/X-HPI/O2 systems.
and 3-F), whereas 4-MeO-HPI gives a yield more than double
that of the unsubstituted HPI, and also decidedly higher than
those with HBT and VLA.
This result can be rationalised considering that the major
contribution to the overall reaction rate is given by the oxida-
tion of the aryl-substituted HPI to the N-oxyl radical (X-
PINO) by laccase (Scheme 5, step a). This process should be
favoured by electron-donor X-substituents that lower the
mediator redox potential (cf. data in Tables 1 and 2).26b On
the other hand, the H-abstraction process between p-anisyl
alcohol and the X-PINO (Scheme 5, step b) should be favoured
by electron-withdrawing X-substituents that give rise to stron-
ger NO–H bonds (in Table 2).26b
Since 4-MeO-HPI emerges as the most efficient mediator,
not only among the X-HPIs but even considering all those
investigated here (excluding TEMPO and its derivates), it has
been chosen for appraising the selectivity of the aerobic
oxidation (see below).
The effect of these substituents on the mediation efficiency by
the HPIs towards laccase has been assessed here in the bench-
mark oxidation (Scheme 2). The reactions were run at room
temperature for a shorter (15 h) time, in order to better
discriminate these mediators in efficiency. The buffered water
solution (0.1 M sodium citrate, pH 5.0), previously purged with
O2, contained 25% dioxane as a co-solvent for better solubility
of the substituted HPIs. Once again, the only oxidation
product observed in all cases was p-anisaldehyde, the rest of
mass balance being the recovered substrate. The results are
given in Table 2 and compared with those obtained (under the
same conditions, i.e., 15 h) with two of the best mediators of
Table 1, namely HBT and VLA.14 Available BDENO–H and
redox data of the mediators are listed,26b,39 to help in the
interpretation.
Selectivity between lignin and polysaccharides
In a search for new oxidation strategies that take place in an
environmental friendly way in water solution,40 and might
enable a selective degradation of lignin in wood pulp for paper
manufacture,2,4,7,41 4-MeO-HPI has been evaluated as the
mediator of laccase in the oxidation of two model compounds
of woody tissue. These were a dimeric model of lignin (1),15a,42
having a secondary benzylic alcohol residue, and a dimeric
model of hemicellulose (2),43 which is believed to ‘tie’ lignin to
cellulose.11 Oxidation of a 1 : 1 mixture of these two models
with laccase and 4-MeO-HPI in the presence of O2 was
performed at room temperature for a 24 h reaction time
(Scheme 6).
Our system oxidised selectively model 1 to the carbonyl
derivative 3 (58% yield, referred to the initial amount of 1),
whereas it did not oxidise the aliphatic alcohol groups present
in model 2, which was recovered unchanged (see Experimental
Section). The lower BDE of the benzylic C–H bonds (ca. 83–87
kcal molꢀ1),19 when compared to that of the aliphatic C–H
bonds (ca. 94–98 kcal molꢀ1),19 explains why this {N–Od
A clear trend of increasing oxidation efficiency on increasing
the electron-richness of the HPIs emerges in Table 2. In fact,
negligible or very low yields of p-anisaldehyde are obtained
with the electron-withdrawing substituted HPIs (4-CH3OCO
Table 2 Yields of p-anisaldehyde in the oxidation of p-anisyl alcohol
by laccase/mediator/O2 systems in buffered water solution (0.1 M
sodium citrate, pH 5), with 25% dioxane as co-solvent, for 15 h at
room temperature
4-MeO-C6H4CHO
yield (%)a
BDENOꢀH
/
E1/V vs.
Mediator
kcal molꢀ1b
NHE in H2Ob
4-CO2Me-HPI
3-F-HPI
HPI
2.0
3.0
13
88.9
88.6
1.18
—
radical (BDEO–H of 4-MeO-HPI ¼ 87.3 kcal molꢀ1 26b
)
ab-
stracts the weaker benzylic C–H bonds geminal to the OH
bond in 1, but not the stronger aliphatic C–H bond geminal to
the OH bond in 2. We have therefore a highly selective HAT
process, endowed with interesting potential applications in the
pulp and paper industry. In contrast, the most efficient med-
iator of laccase in the oxidation of alcohols, i.e., TEMPO,44 is
not suitable for use in the industrial biodelignification because
it lacks selectivity. It oxidises not only benzylic but even
aliphatic alcohols,24 in view of its oxidation mechanism that
88.1
88.2
1.09
—
4-Me-HPI
4-MeO-HPI
HBT
24
33
87.3
(cf. ref. 20)
ca. 80–82c
0.99
1.08
0.92
16
19
VLA
a
The yields % are referred to the initial amount of substrate, and are
the average of three determinations (by GC); typical error: ꢁ3%.
b
c
Refs. 26b and 39. Ref. 19c.
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7
1313

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Scheme 8 Thermochemical cycle for determining BDENO–H values.
original benzyl radical intermediate (4d). Conclusive evidence
for the occurrence of the radical HAT route is thereby pro-
vided. It is apt to remind that oxidation of a similar cyclisable
probe with laccase/TEMPO failed to produce a rearranged
product;23 in fact, no radical intermediate is expected in the
ionic route of Scheme 4.24
Scheme 6 Competitive oxidation of a lignin model (1) and of a
polysaccharide model (2).
Bond energies from a thermochemical cycle and theoretical
calculation
is ionic and not radical, and consequently oxidises even the
carbohydrate component of wood, as already demonstrated.45
On the basis of the approach delineated in a seminal Accounts
paper,49 we have exploited a thermochemical cycle in order to
have access to the BDENO–H value of {NO–H mediators that
are not experimentally accessible by means of the EPR equili-
bration method.19b,c The cycle is given in Scheme 8, and
Intercepting the radical intermediate
50
requires the knowledge of pKa and E114 data.
The radical HAT route of oxidation available to {N–OH
mediators (Scheme 3) requires H-abstraction from the benzylic
position of a non-phenolic substrate, with the consequent
formation of a benzyl radical intermediate.13,46 Further reac-
tion with dioxygen leads to the observed carbonyl end-pro-
ducts.26b Capture of an intermediate in a multistep reaction is a
classic clue for elucidating a mechanism, but no attempts to
intercept the benzyl radical intermediate of the HAT oxidation
with laccase/{N–OH mediators have ever been made. We
wanted to acquire this particular evidence, in addition to the
other evidence already consistent with the HAT route.13
A suitable probe substrate was foreseen in 2-allyloxybenzyl
alcohol (4), in keeping with the radical-clock strategy.47 The
expected oxidation product of 4 is the corresponding 2-allyloxy-
benzaldehyde (5), but the intermediate benzyl radical (4d)
could be diverted and trapped in a fast 6-membered ring
formation according to an intramolecular 6-exo-trig process
(kc),48 with ensuing functionalisation (Scheme 7).
Both pKa and E1 values have the dimensions of free energies,
whereas an entropy correction must be applied to BDE (an
enthalpic value), and also the solvation of the H1/Hd couple
must be taken into account:49 these details are dealt with in the
above paper.49 We have calibrated the cycle in the case of HPI,
for which pKa, E11 and BDE data are all known (Table 3), in
order to confine and extract both the entropy correction and
the solvation term in the E21 parameter, which we then use in
the calculation of the BDENO–H for the other structurally
similar {NO–H mediators.
From our calculations it clearly emerges that all of these
{N–OH mediators have a BDENO–H value above 80 kcal
mol ,
ꢀ1 20 thereby sufficient for making the H-abstraction step in
Scheme 3 possible. These numbers support the relative profi-
ciency of mediation given in Table 1 and, in particular, HBT
comes out efficient even from a thermochemical viewpoint.
Determination of the BDENO–H of HBT has been also
accomplished by quantum chemical calculations (Gaussian
98 package)51 by the Density Functional approach (DFT).
The geometries of the starting neutral HBT and of the corre-
sponding N–Od radical were optimized at the MPW1P86/6-
311G**level. This functional was chosen because it is reported
to yield the best agreement with experimental BDEs.52
Precursor 4 was synthesised in an uneventful route (see
Experimental), and subjected to oxidation with laccase/HBT
under the usual conditions. As projected, the formation of
aldehyde 5 was accompanied by a product that MS and NMR
analyses showed to be the 4-chromanone derivative 6. This is
the reasonable outcome of further oxidation of the cyclic
radical (c-4d), deriving from the 6-exo-trig ring-closure of the
Table 3 Relevant data for the calculation of BDENO–H values (see
Scheme 8). Shortened names of the {NO–H mediators are from Table
1. In brackets, the values of pKa and E11 are converted in kcal molꢀ1 by
multiplying by 1.4 and 23.06, respectively
BDENO–H
/
pKa in H2Oa
E11
E12
kcal molꢀ1
b
c
{NO–H
HPI
6.3 (8.8)
4.6 (6.4)
4.3 (6.0)
4.0 (5.6)
3.8 (5.3)
3.5 (4.9)
1.09 (25.1)
1.08 (24.9)
0.92 (21.2)
1.2 (27.7)
(54)
(54)
(54)
(54)
(54)
(54)
88.1d
85e
HBT
VLA
HBO
TFBT
HOAT
a
82e
85–87ef
83–85ef
83–85ef
1.11 (25.6)
1.14 (26.3)
From ref. 50 (value in kcal molꢀ1 in brackets). From Table 1 (value
b
in kcal molꢀ1 in brackets). In kcal molꢀ1; obtained from Scheme 8 for
c
HPI (ref. 49 gives 53 kcal molꢀ1). Experimental value (ref. 26b).
d
e
Obtained from Scheme 8 as: BDE ¼ (pKa þ E11 þ E12), all data being
in kcal molꢀ1
.
Uncertainty due to the solvent (MeCN instead of H2O)
f
Scheme 7 Intercepting the radical intermediate of the HAT oxidation
mechanism.
used in the E11 determination.
1314
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View Article Online
Harmonic vibrational frequencies were calculated at the same
level of theory to confirm that the optimized structures corre-
spond to local minima, and to determine zero-point energy
(ZPE) corrections. The BDENO–H of HBT results in this way to
be 85.9 kcal molꢀ1, in excellent agreement with the value (85
kcal molꢀ1) obtained from the thermochemical cycle. This
approach has been validated by back-calculating the experi-
mental BDENO–H value of HPI (i.e., 88.1 kcal molꢀ1), and a
very satisfactory result of 89.7 kcal molꢀ1 obtained.
example a-monodeuteriated p-MeO-benzyl alcohol,13 1-(3,4-
dimethoxyphenyl)-2-phenoxy-1-ethanol (1) and 1-(3,4-di-
methoxyphenyl)-2-phenoxy-1-ethanone (3),15 while 2-allyloxy-
benzaldehyde (5) is commercial (Aldrich). The seven-step
synthesis of methyl-3-O-(a-L-arabinofuranosyl)-b-D-xylopira-
noside (2), starting from methyl-b-D-xylopiranoside and
methyl-a-D-arabinofuranoside, is from the literature.43 We
have repeated it obtaining 2 in an overall 3% yield, and
NMR characterisation of 2 is consistent with the one pub-
lished.43 The synthesis of a few mediators (entries 3,4,12,19 in
Table 1) had been described elsewhere.19b,53 The substituted
N-hydroxyphthalimides used in Table 2 were synthesised as
reported before.26b Acetonitrile was dried over molecular sieves
(4 A) for at least 24 h under Ar prior to the electrochemical
experiments. Commercial Bu4NBF4 (Fluka) was crystallised
from ethyl acetate–petroleum ether, and dried under vacuum
(10ꢀ2 Torr) at 60 1C for 6 h; commercial anhydrous LiClO4
(Aldrich) was used as received. Buffers were prepared using
ultrapure water obtained from a MilliQ apparatus.
Conclusions
A number of new potential mediators of laccase has been
comparatively evaluated and ranked towards the benchmark
aerobic oxidation of p-MeO-benzyl alcohol. The mechanism of
oxidation of this non-phenolic substrate by each mediator has
been assessed among three alternatives. An ionic mechanism
results viable to persistent {N–Od species (such as TEMPO)
that can be converted into the {N O1 form (i.e., Medox) on
Q
monoelectronic oxidation at a redox potential accessible to
laccase, and therefore not exceeding 1 V. A radical H-abstrac-
tion mechanism (the HAT route) is instead followed by {N–
OH mediators that laccase oxidatively converts into short-lived
{N–Od intermediates, provided that the energy of the NO–H
bond they give rise to on abstracting H-atom from the sub-
strate is at least 80 kcal molꢀ1 or higher. A redox mechanism
may be finally accessible to species, such as ABTS, that are
easily oxidised by laccase.30c Experimental characterisation of
some of the mediators, by means of spectrophotometric,
electrochemical and thermochemical survey, supports these
assessments. Very significant is the finding that catechol, a
model of phenolic moieties of lignin, once oxidised by laccase
performs as a radical mediator in the oxidation of non-
phenolic groups. This supports a stand alone role as delignify-
ing enzyme for laccase in nature, without the need for added
mediators. Clear-cut evidence for the formation of a benzyl
radical intermediate in the HAT oxidation of a benzyl alcohol
by laccase and {N–OH mediators is gathered through a
trapping experiment with a cyclisable probe. The selectivity
of the laccase-catalysed oxidation of competing lignin and
polysaccharide model compounds has been determined by
using the highly proficient 4-MeO-HPI mediator, and found
very high in favour of the former model. This is in keeping with
the operation of a radical HAT process that efficiently cleaves
the benzylic C–H bond with respect to the aliphatic C–H bond
of the two models. The selective HAT oxidation by laccase/
{N–OH mediators shows therefore interesting promises for
application in the pulp and paper industry. Finally, an evalua-
tion of the BDENO–H value of HBT, which is not accessible
experimentally, is provided by the use of a thermochemical
cycle, and the approach is extended to other {N–OH media-
Synthesis of 2-allyloxybenzyl alcohol (4)
2-Hydroxybenzyl alcohol (Aldrich; 3.2 g, 25 mmol), dissolved
in 5 mL of acetone, was allylated with 2.2 mL of allyl bromide
(25 mmol) in the presence of 3.6 mg of K2CO3 (26 mmol) under
stirring for 14 h at room temperature. Acetone was removed,
the residue taken-up in diethyl ether, extracted with Na2CO3
solution and dried (an. Na2SO4). Removal of the solvent, and
filtration over a short-path silica gel column, gave 4 in 50%
yield; m/z 164. 1H NMR (200 MHz, CDCl3) dH 2 (bs, 1H, OH),
4.55–4.50 (d, 2H, OCH2), 4.65 (s, 2H, CH2OH), 5.45–5.25
(ddq, 2H, CH ), 6.1–5.9 (m, 1H, CH ), 7.3–6.8 (m, 4H,
Q
Q
2
ArH); 13C NMR (50 MHz, CDCl3) dC 62 (CH2OH), 69
(ArOCH2), 117 (CH ), 111–127 (CAr-H), 129 (CAr
–
Q
2
CH OH), 133 (CH CH ), 156 (CAr–OCH2).
Q
2
2
Enzyme purification
Laccase from a strain of Trametes villosa (viz. Poliporus
pinsitus) (Novo Nordisk Biotech) was employed. It was pur-
ified by ion-exchange chromatography on Q-Sepharose by
elution with phosphate buffer, and laccase fractions having
an absorption ratio A280/A610 of 20–30 were considered suffi-
ciently pure.54 The collected fractions were concentrated by
dialysis in cellulose membrane tubing (Sigma) against poly
(ethylene glycol) to a final activity of 8000 U mLꢀ1, as
determined spectrophotometrically by the standard assay with
ABTS.29
Enzymatic reactions
tors. DFT calculations do confirm this result, as 85 kcal molꢀ1
.
The aerobic oxidations reported in Table 1 were performed at
room temperature in stirred water solution (3 mL), buffered at
pH 5 (0.1 M in sodium citrate) and purged with O2 for 30 min
prior to the addition of the reagents.13,14 In general, the initial
concentration of the reagents was: [p-anisyl alcohol] 20 mM,
[mediator] 6 mM, with 3 U mLꢀ1 of laccase, and the reaction
time was 24 h at room temperature, an atmosphere of oxygen
being kept in the reaction vessel by means of a hemi-inflated
latex balloon. The oxidation reactions reported in Table 2 were
similarly performed at room temperature, but a 4 : 1 buffered
water : dioxane mixed solvent (5 mL) was employed to ensure
full solubility of the HPIs mediators. The initial concentrations
were: [p-anisyl alcohol] 5 mM, [mediator] 1.7 mM, with 1 U
mLꢀ1 of laccase, and the reaction time was shorter (15 h) than
in Table 1. The reaction mixture was extracted with ethyl
acetate or with CH2Cl2, dried over Na2SO4 and characterised
by GC-MS. The yields of oxidation (Table 1 and 2) were
reckoned by GC analysis from multiple injections with respect
to an internal standard (acetophenone or 4-methoxyacetophe-
Experimental
General remarks
NMR spectra were taken on a AC 300 Bruker instrument.
EPR spectra were recorded on a Bruker ESP 300 spectrometer.
A VARIAN CP 3800 GC, fitted with a 30 m ꢂ 0.25 mm methyl
silicone gum capillary column (CPSil5CB), was employed in
the analyses. The identity of the products was confirmed
by GC-MS analyses, run on a HP 5892 GC, equipped with a
30 m ꢂ 0.2 mm methyl silicone gum capillary column, and
coupled to a HP 5972 MSD instrument, operating at 70 eV.
Materials
Most of the mediators or substrates were commercially avail-
able (Aldrich) or already present in the laboratory,13,14,21 as for
N e w J . C h e m . , 2 0 0 5 , 2 9 , 1 3 0 8 – 1 3 1 7
1315

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none or 4-methylbenzophenone), using suitable response
factors determined from authentic products. A good material
balance (495%) was observed in all the experiments. In the
absence of the mediator or the enzyme, no products were
observed in significant amounts (o0.1%).
acetate buffer containing 0.1 M LiClO4. A three-electrode
circuit was used with either an electrochemical computerized
system (Amel System 5000, version 2.1), or a potentiostat with
positive feedback ohmic-drop compensation,55 and a MPLI
Hardware (Vernier Software & Technology) controlled by a
program written in Cþþ language for Windows 95/98.56 The
auxiliary electrode was a platinum wire (surface 1 cm2), and
either a saturated calomel electrode (SCE) or a Ag/AgCl/KCl 3
M electrode was used as reference. Redox potential values are
referred to NHE. The scanning rate ranged from 0.005 V sꢀ1 to
50 V sꢀ1. The investigated mediator was 1–2 mM, and a
twofold excess of pyridine in MeCN was used to deprotonate
the {N–OH mediators into the more easily oxidisable
{N–Oꢀ form (see pKa values in Table 3).
Competitive aerobic oxidation of model compounds 1 and 2
Mediator 4-MeO-HPI (18 mmol), laccase (20 units) and 40
mmol of each of the models 1 and 2 were added to 6 mL of a
buffered solution (0.1 M sodium citrate, pH 5.0) with 5%
MeCN as co-solvent, which had been purged with O2 for 30
min before the addition of the reagents. The mixture was
magnetically stirred at room temperature for 15 h under
oxygen (filled balloon), and then worked-up with ethyl acetate.
The solvent was removed under reduced pressure, the reaction
mixture acetylated with Ac2O in pyridine and finally analysed
by 1H NMR. Model 1 underwent selective oxidation to 1-(3,4-
dimethoxyphenyl)-2-phenoxy-1-ethanone (3) (58% yield, re-
ferred to the initial amount of 1). 39% of 1 was recovered as
the acetyl derivative (referred to the starting material, and
calculated from the integrated signal of the benzylic CHOAc at
6.09 ppm). 96% of model 2 was recovered as the per-acetylated
derivative (the recovery is referred to the starting material, and
calculated from the integrated signal of the H-1ara at 5.09
ppm). 1H NMR (CDCl3, 200 MHz) d 1.91–2.20 (s, 15H,
COCH3), 3.30 (dd, J ¼ 5.0, 10.5, 1H, H-5xyl), 3.78 (m, 1H,
H-5xyl), 4.05 (m, 1H, H-3xyl), 4.20 (m, 1H, H-4ara), 4.25 (m, 2H,
H-5ara), 4.28 (d, J ¼ 7.5, 1H, H-1xyl), 4.85 (m, 1H, H-2xyl), 4.85
(m, 1H, H-4xyl), 4.81–4.90 (m, 2H, H-2ara and H-3ara), 5.09
(s, 1H, H-1ara).
Acknowledgements
We thank Prof. Marco Lucarini (University of Bologna, Italy)
for taking an EPR spectrum. Thanks are also due to Novo
Nordisk Biotech (Denmark) for a generous gift of laccase, to
the EU for a grant (QLK5-CT-1999-01277), and to the Italian
MIUR for financial support (COFIN and FIRB).
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Cyclic voltammetry at the steady disc electrode (glassy-carbon
disc, 1.5 mm in diameter)14 was carried out at 25 1C in MeCN
containing 0.1 M Bu4NBF4, or alternatively in 0.01 M sodium
1316
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